CN107406889A - By injecting the selection based on drop carried out - Google Patents

By injecting the selection based on drop carried out Download PDF

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CN107406889A
CN107406889A CN201680016459.8A CN201680016459A CN107406889A CN 107406889 A CN107406889 A CN 107406889A CN 201680016459 A CN201680016459 A CN 201680016459A CN 107406889 A CN107406889 A CN 107406889A
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bacillus
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polynucleotides
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A·法拉阿拉吉
O·许格博基格
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Novo Nordisk AS
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    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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Abstract

A kind of method for being used for the polynucleotides of identification code enzyme interested in microfluidic devices, this method are carried out by the following：The emulsion of the microfluid drop in the library comprising the polynucleotides for encoding one or more enzymes is provided, it is introduced into allow to expand polynucleotides PCR to the concentration PCR solution in the drop of selection and/or by lethal solution and is introduced into the drop for cancelling selection, and the polynucleotides of identification code enzyme interested.

Description

By injecting the selection based on drop carried out
Technical field
The present invention relates to a kind of microfluid or " chip lab " method.More specifically, it relates to how to use fluid Inject as a kind of means that selection water droplet is selected or cancelled in immiscible carrier fluid.Water droplet is sometimes referred to as droplet Or microcapsules, they generally have about 20 microns of average diameter, and are used as compartment or small reaction vessel.They can With the viable microbial cells containing such as secretase.Drop can also contain other components, such as fluorescent enzyme substrate, the luciferase Substrate can show the activity of the enzyme as caused by the microbial cell in drop.
Background technology
One of separation coding with desired active gene outcome is described at 1999 (WO 99/02671) Kind or a variety of genic universals, it comprises the following steps：(a) gene is separated into microcapsules；(b) expression is lost The factor is passed to produce their own gene outcome in microcapsules；(c) sorting, which produces, has desired active gene The gene of product.
Have been described and be based on silicone using the vitro expression systems in microcapsules and the chemically inert of stable emulsifying Surfactant (WO 03/044187).
Such as by applying electric field, the manipulation of droplet is had been realized in, include merging or the coalescence (WO of several drops 2007/089541)。
Many comprehensive surveys can be obtained, and many microfluid components are all commercially available.Microfluidic field is fast Speed development, and any potential improve all is highly desirable to.
The content of the invention
The inventor of this method is it has been shown that the identification for encoding the polynucleotides of enzyme interested can be in microfluidic device In effectively carried out by the following：The microfluid in the library comprising the polynucleotides for encoding one or more enzymes is provided The emulsion of drop, concentration PCR solution is introduced, it allows polynucleotides PCR amplifications into the drop of selectionAnd/orWill be lethal molten Liquid is introduced into the drop for cancelling selection, then the polynucleotides of identification code enzyme interested.
Correspondingly, the invention provides the method for the polynucleotides of the enzyme interested of identification code in microfluidic devices, It the described method comprises the following steps：
A) emulsion of the microfluid drop in the library comprising the polynucleotides for encoding one or more enzymes is provided, wherein each Drop includes the at most single member in library, and expresses the libraries of polynucleotides to produce one or more enzymes；
B) aliquot of the substrate comprising one or more enzymes is introduced into each drop, one or more of which sense is emerging The presence of the organized enzyme of interest changes into substrate the product that can be screened；
C) determine anti-containing the positive drop for screening product for being higher than predetermined threshold level, the predetermined threshold level Come over and determine negative drop；
D) aliquot of the concentration PCR solution comprising nucleotides, suitable archaeal dna polymerase and one group of PCR primer is introduced, It allows to arrive the polynucleotides PCR amplifications for encoding one or more enzymes interested into each positive determined in step (c) In drop；And/orDNA will be caused to be denatured and the aliquot of enzyme denaturation and/or the solution of cell cracking is introduced in step (c) In the negative drop of middle determination；
E) polynucleotides of clone or the one or more enzymes interested of PCR amplification codings from positive drop；With
F) from the polynucleotides of the enzyme interested of the polynucleotides identification code in the middle clone of step (e) or PCR amplifications.
Definition
Coded sequence：Term " coded sequence " means the polynucleotides of the amino acid sequence of directly specified polypeptide.Code sequence The border of row is typically determined that the open reading frame is since initiation codon (such as ATG, GTG or TTG) by open reading frame And terminated with terminator codon (such as TAA, TAG or TGA).Coded sequence can be genomic DNA, cDNA, synthetic DNA or its Combination.
Control sequence：The polynucleotides for the mature polypeptide that term " control sequence " means to encode the present invention for expression must The nucleotide sequence needed.Each control sequence can be natural (that is, from identical for the polynucleotides for encoding the polypeptide Gene) or external source (that is, from different genes), or be natural or external source relative to each other.Such control sequence includes But it is not limited to targeting sequencing, polyadenylation se-quence, propeptide sequence, promoter, signal peptide sequence and transcription terminator.Extremely Few, control sequence includes promoter and transcription and translation termination signal.Be advantageous to for introducing by these control sequences with compiling The purpose of the specific restriction enzyme enzyme site of the code area connection of the polynucleotides of code polypeptide, these control sequences can be provided with Multiple joints.
Expression：Term " expression " includes being related to any step caused by polypeptide, including but not limited to transcribes, is repaiied after transcription Decorations, translation, posttranslational modification and secretion.
Expression vector：Term " expression vector " means wire or ring-shaped DNA molecule, and the molecule includes the multinuclear of coded polypeptide The control sequence that thuja acid and the polynucleotides are operationally used for its expression with providing is connected.
Host cell：Term " host cell " means to be easy to the nucleic acid construct or table with the polynucleotides comprising the present invention Up to any cell type of carrier conversion, transfection, transduction etc..The term " host cell " covers the spawn of parental cell, Due to the mutation occurred during duplication, the offspring and its parental cell are incomplete same.
Separation：Term " separation " means the material being in nature in the form being not present or environment.Separation The non-limiting examples of material include (1) any non-naturally occurring material, and (2) include but is not limited to any enzyme, variant, core Acid, protein, any material of peptide or co-factor, the material is at least in part from the one or more with its this qualitative correlation or institute Have in naturally occurring composition and remove；(3) manually modified any material is passed through relative to the material naturally found；Or (4) are logical Cross relative to its natural related other components, any material for increasing the amount of material and modifying (such as in host cell Restructuring produces；Encode multiple copies of the gene of the material；And opened using natural more related than the gene to encoding the material The stronger promoter of mover).
Nucleic acid construct：Term " nucleic acid construct " means single-stranded or double-stranded nucleic acid molecules, and the nucleic acid molecules are from day So separated in existing gene, or be modified to include the section of nucleic acid in a manner of being not present in nature originally, or It is synthesis, the nucleic acid molecules include one or more control sequences.
It is operably connected：Term " being operably connected " means the coded sequence relative to polynucleotides by control sequence Placement so causes the control sequence to instruct the configuration of the expression of the coded sequence in position.
Sequence identity：Relevance between two amino acid sequences or between two nucleotide sequences passes through parameter " sequence identity " describes.
For purposes of the present invention, using such as in EMBOSS program bags (EMBOSS:The European Molecular Biology Open Software Suite[EMBOSS：European Molecular Biology Open software suite], Rice et al., 2000, Trends Genet. [science of heredity trend] 16:276-277) institute is real in your program of the Maimonides of (preferably 5.0.0 versions or more redaction) (Needleman and Wunsch, 1970, J.Mol.Biol. [divide Ned Coleman-wunsch (Needleman-Wunsch) algorithm applied Sub- biology magazine] 48:443-453) determine the sequence identity between two amino acid sequences.Used parameter is empty The open point penalty 10 in position, gap extension penalties 0.5, and EBLOSUM62 (BLOSUM62 EMBOSS versions) substitution matrix.Will mark (acquisition of use-non-reduced (nobrief) option), which is exported, for the Maimonides of " most long uniformity " your (Needle) is used as percentage one Cause property and be calculated as below：
(consistent residue x 100)/(comparing the room sum in length-comparison)
For purposes of the present invention, using such as in EMBOSS program bags (EMBOSS:The European Molecular Biology Open Software Suite[EMBOSS：European Molecular Biology Open software suite], Rice et al., 2000, See above) Ned Coleman-wunsch algorithm for being implemented in the Maimonides of (preferably 5.0.0 versions or more redaction) your program (Needleman and Wunsch, 1970, see above) determine the sequence identity between two deoxyribonucleotide sequences. Used parameter is Gap Opening Penalty 10, gap extension penalties 0.5, and EDNAFULL (NCBI NUC4.4 EMBOSS Version) substitution matrix.The Maimonides of " most long uniformity " your (Needle) will be labeled as and export (use-non-reduced (nobrief) option Obtain) it is used as Percent Identity and is calculated as below：
(consistent deoxyribonucleotide x 100)/(comparing the room sum in length-comparison)
Detailed description of the Invention
In a first aspect, the present invention relates to the polynucleotides of the enzyme interested for identification code in microfluidic devices Method, it the described method comprises the following steps：
A) emulsion of the microfluid drop in the library comprising the polynucleotides for encoding one or more enzymes is provided, wherein each Drop includes the at most single member in library, and expresses the libraries of polynucleotides to produce one or more enzymes；
B) aliquot of the substrate comprising one or more enzymes is introduced into each drop, one or more of which sense is emerging The presence of the organized enzyme of interest changes into substrate the product that can be screened；
C) determine anti-containing the positive drop for screening product for being higher than predetermined threshold level, the predetermined threshold level Come over and determine negative drop；
D) aliquot of the concentration PCR solution comprising nucleotides, suitable archaeal dna polymerase and one group of PCR primer is introduced, It allows to arrive the polynucleotides PCR amplifications for encoding one or more enzymes interested into each positive determined in step (c) In drop；And/orDNA will be caused to be denatured and the aliquot of enzyme denaturation and/or the solution of cell cracking is introduced in step (c) In the negative drop of middle determination；
E) polynucleotides of clone or the one or more enzymes interested of PCR amplification codings from positive drop；With
F) from the polynucleotides of the enzyme interested of the polynucleotides identification code in the middle clone of step (e) or PCR amplifications.
In a preferred embodiment of the invention, enzyme interested be hydrolase, isomerase, ligase, lyases, oxidation also Protoenzyme or transferase；Preferably, the enzyme interested be a kind of alpha-galactosidase, alpha-Glucosidase, aminopeptidase, amylase, Asparaginase, beta galactosidase, β-glucosyl enzym, xylobiase, carbohydrase, carboxypeptidase, catalase, cellobiose Hydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyl transferases, deoxyribonuclease, endoglucanase, Esterase, green fluorescent protein, glucanotransferase, glucoamylase, invertase, laccase, lipase, mannosidase, change are poly- Carbohydrase, oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribalgilase, turn Glutaminase or zytase.
In yet another preferred embodiment, the library encodes one or more hydrolases, isomerase, ligase, cracking Enzyme, oxidoreducing enzyme or transferase；Preferably, the library encodes one or more alpha-galactosidases, alpha-Glucosidase, ammonia peptide Enzyme, amylase, asparaginase, beta galactosidase, β-glucosyl enzym, xylobiase, carbohydrase, carboxypeptidase, hydrogen peroxide It is enzyme, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyl transferases, deoxyribonuclease, interior Cut dextranase, esterase, green fluorescent protein, glucanotransferase, glucoamylase, invertase, laccase, lipase, sweet dew Glycosidase, become dextranase, oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, core Ribonuclease T., transglutaminase or zytase.
In a preferred embodiment, the different variants of library coding same enzyme, and in another embodiment, this article Storehouse includes two or more different polynucleotides of coding same enzyme.
It is contemplated that the method for the present invention can use the polynucleotides of separation and the library of vitro expression systems.However, The preferred embodiments of the present invention are to set in vivo, and wherein the library is included in prokaryotic host cell；It is preferred that in Gram-positive In host cell；More preferably in bacillus (Bacillus) host cell；Even more preferably in Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus Clausii), bacillus coagulans (Bacillus coagulans), bacillus firmus (Bacillus firmus), brilliance Bacillus (Bacillus lautus), bacillus lentus (Bacillus lentus), bacillus licheniformis (Bacillus Licheniformis), bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus Pumilus), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus subtilis (Bacillus Subtilis) or in bacillus thuringiensis (Bacillus thuringiensis) host cell；And most preferably in lichens In Bacillus host cell.
Naturally, each single polynucleotides in library are in the single host cell of their own.Correspondingly, preferred real Apply in example, each drop the first aspect the step of in (a) includes most single host cells.
Enzyme product that is detectable or can quantifying is converted into qualitatively or quantitatively to determine by multinuclear by detecting zymolyte The activity of one or more enzymes of thuja acid library coding.Generally, fluorescent enzyme substrate is added, is become to be detected or measurement glimmering Light enzyme product.
Therefore, in a preferred embodiment of the invention, the substrate for one or more enzymes is fluorescence, and the work of enzyme Fluorogenic substrate is converted into fluorescence-causing substance by property.
Once enzymatic activity of special interest is detected in selected drop, it is necessary to more in drop to be identified Nucleic acid libraries member.Generally, polynucleotides are identified by DNA sequencing.
When building library, polynucleotides may be fitted with identifying sequence label for use as " bar code ", so as to exclude The needs of sequencing.Identification based on bar code, will immediately know that the DNA sequence dna of polynucleotides, therefore be identified.
The present invention preferred aspect, by the DNA sequencing of polynucleotides in step (f) identification code enzyme interested Polynucleotides.
In the first aspect of the present invention, several aliquots of solution are introduced into drop.The volume of aliquot is usual It is more much smaller than drop, but their size in principle can be equal with the volume of drop even more big.In the following example, etc. Divide sample significantly less than drop.Aliquot is introduced into drop in microfluidic devices or said two drops in another way Merge or the method for coalescence there are many kinds.
Such as disclosed in WO 2007/061448 and can apply electric field to merge or coalesce two or more drops Microfluidic device design.Another method that the small aliquot of waterborne liquid is introduced into water droplet in microfluidic devices It is referred to as " micro-injection ", and has disclosure in such as WO 2010/151776.
In the following example, aliquot and droplet coalescence or coalescence are introduced aliquot by applying electric field In drop.
Correspondingly, in the preferred embodiment of first aspect, by applying electric field or by injecting aliquot and liquid Drop merges or aliquot is incorporated into drop by coalescence.
Polynucleotides
The invention further relates to the library for the polynucleotides for encoding one or more enzyme polypeptides as described herein.
Technology for separating or cloning polynucleotides is well known in the art, and including from genomic DNA or CDNA, or its combination separation.Can be for example by using well known polymerase chain reaction (PCR) or the antibody screening of expression library To detect the cloned DNA fragments with apokoinou construction feature, realize from genomic dna cloning polynucleotides.See, for example, Innis et al., 1990, PCR:A Guide to Methods and Application[PCR：Methods and applications guide], Academic Press [academic press], New York.Other amplification procedures such as ligase chain reaction can be used (LCR) activated transcription (LAT) and the amplification (NASBA) based on polynucleotides, are connected.
Encoding the modifications of the polynucleotides of polypeptide of the present invention the polypeptide of the polypeptide is substantially similar to for synthesis to be It is required.Term " is substantially similar to " the non-naturally occurring form that the polypeptide refers to the polypeptide.These polypeptides can be because of certain Kind of engineered way and the polypeptide from being separated from its natural origin is different, such as in specific activity, heat endurance, optimal pH etc. Different variants.Variant can be constructed as below：It is more based on being proposed as mature polypeptide encoded sequence (for example, its subsequence) Nucleotides, and/or by introducing nucleotide subsitution, the nucleotide subsitution does not cause the amino acid sequence of polypeptide to change, still Used corresponding to the codon for being intended to the host organism for producing enzyme, or different aminoacids sequence can be produced by introducing Nucleotide subsitution.For the general description of nucleotides substitution, see, e.g. Ford et al., 1991, Protein Expression And Purification [protein expression and purifying] 2:95-107.
Nucleic acid construct
The invention further relates to nucleic acid construct, these nucleic acid constructs include being operably coupled to one or more controls The polynucleotides of the invention of sequence, under conditions of compatible with control sequence, this or these control sequence instructs code sequence The expression being listed in suitable host cell.
Can be with polynucleotides described in multi-mode operation perhaps in order to the expression of polypeptide.Depending on expression vector, in multinuclear It can be desirable or required that manipulation is carried out to it before thuja acid insertion vector.For being modified using recombinant DNA method The technology of polynucleotides is well known in the art.
The control sequence can be promoter, i.e. be identified by host cell with the polynucleotides to encoding polypeptide of the present invention The polynucleotides expressed.The promoter includes transcriptional control sequence, and these sequences have mediated the expression of the polypeptide.The startup Son can be any polynucleotides that transcriptional activity is shown in host cell, including saltant type, truncated-type and heterozygous open Mover, and can be obtained by encoding with homologous or heterologous extracellular or intracellular polypeptides the gene of the host cell.
The example of suitable promoter for the transcription for the nucleic acid construct that the present invention is instructed in bacterial host cell is The promoter obtained from following gene：Bacillus amyloliquefaciens alpha-amylase gene (amyQ), bacillus licheniformis alpha-starch Enzyme gene (amyL), bacillus licheniformis penicillinase gene (penP), bacillus stearothermophilus production maltogenic amylase base Because of (amyM), subtilis levansucrase gene (sacB), bacillus subtilis xylA and xylB gene, Su Yunjin Bacillus cryIIIA genes (Agaisse and Lereclus, 1994, Molecular Microbiology [molecular microbiology] 13:97-107), E. coli lac operon, Escherichia coli trc promoters (Egon et al., 1988, Gene [genes] 69: 301-315), streptomyces coelicolor agarase gene (dagA) and protokaryon beta-lactam enzyme gene (Villa-Kamaroff Et al., 1978, Proc.Natl.Acad.Sci.USA [NAS's proceedings] 75:3727-3731) and tac starts Sub (DeBoer et al., 1983, Proc.Natl.Acad.Sci.USA [NAS's proceedings] 80:21-25). Gilbert et al. (1980) Scientific American [science American], 242:" Useful in 74-94 In proteins from recombinant bacteria " [the useful proteins matter from recombinant bacteria]；And Sambrook et al. describes other promoter in (1989, see above).The example of Gene expression is disclosed in WO 99/ In 43835.
Control sequence, which can also be, to be identified by host cell to terminate the transcription terminator of transcription.Terminator is more with encoding this 3 '-end of the polynucleotides of peptide is operably connected.Any terminator of functional can be used for this hair in host cell In bright.
The preferred terminator of bacterial host cell obtains from the gene for the following：Bacillus clausii alkalescence egg White enzyme (aprH), bacillus licheniformis alpha-amylase (amyL) and Escherichia coli rRNA (rrnB).
Control sequence can also be the stable sub-districts of the mRNA of the upstream of coding sequence of promoter downstream and gene, and it increases should The expression of gene.
The example of the stable subregions of suitable mRNA obtains from following：Bacillus thuringiensis cryIIIA genes (WO 94/25612) (Hue et al., [bacteriology is miscellaneous by 1995, Journal of Bacteriology with bacillus subtilis SP82 genes Will] 177:3465-3471).
Control sequence can also be that coding is connected the secretion path for and guiding polypeptide to enter cell with the N- ends of polypeptide The signal peptide coding region of signal peptide.The 5 ' of the coded sequence of polynucleotides-end can be inherently included in translation reading frame in The signal coding sequence that the section of the coded sequence of coded polypeptide natively connects.Alternately, the 5 ' of coded sequence-end It is external signal coding sequence that can include relative to coded sequence.Do not encoded in coded sequence comprising signal peptide natively In the case of sequence, it may be necessary to extraneous signal peptide-coding sequence.Alternately, extraneous signal peptide-coding sequence can be merely Natural signals peptide-coding sequence is substituted to strengthen the secretion of polypeptide.However, it is possible to use guidance has expressed polypeptide and has entered host Any signal coding sequence of the secretory pathway of cell.
Useful signal peptide-coding sequence for bacterial host cell is formed sediment from the Fructus Hordei Germinatus of bacillus NCIB 11837 sugar Powder enzyme, bacillus licheniformis subtilopeptidase A, Di clothing Ya spore Gan Jun Calcium-lactamase, the fatty Ya spores Gan Jun Ru-shallow lake of Shi heat What the gene of powder enzyme, stearothermophilus neutral protease (nprT, nprS, nprM) and bacillus subtilis prsA obtained Signal coding sequence.Simonen and Palva, 1993, Microbiological Reviews [microorganism comment] 57:109- 137 describe other signal peptide.
Control sequence can also be the propeptide code sequence of propetide of the coding at peptide N-terminus.The polypeptide quilt of generation Referred to as preemzyme (proenzyme) or propolypeptide (or being referred to as proenzyme (zymogen) in some cases).Propolypeptide is typically It is inactive and the propetide from propolypeptide can be cut by catalysis cutting or autocatalysis to be converted into active peptides.Can With from bacillus subtilis alkali proteinase (aprE), Bacillus subtilis neutral protease (nprT), thermophilic fungus destroyed wire (Myceliophthora thermophila) laccase (WO 95/33836), rhizomucor miehei (Rhizomucor miehei) day The gene of winter serine protease and cerevisiae alpha-factor obtains propeptide code sequence.
In the presence of signal peptide sequence and propeptide sequence, the propeptide sequence is located immediately adjacent the N- of polypeptide End and the signal peptide sequence are located immediately adjacent the N- ends of the propeptide sequence.
It may also it is desirable to add regulatory sequence, the regulatory sequence regulation is relative to the growth of host cell The expression of polypeptide.The example of regulatory sequence is so that the expression of gene in response to chemical or physical stimulus (including modulating compound Presence) and be turned on and off those.Regulatory sequence in prokaryotic system includes lac, tac and trp operon system.Adjust Other examples of section sequence are those for allowing gene magnification.
Expression vector
The invention further relates to the restructuring of the polynucleotides comprising the present invention, promoter and transcription and translation termination signal Expression vector.Different nucleotides and control sequence can link together to produce recombinant expression carrier, and the recombination expression carries Body can include one or more convenient restriction sites to allow to insert or substitute at these sites to encode the polypeptide Polynucleotides.Alternately, the polynucleotides can be by by the polynucleotides or nucleic acid construct including the polynucleotides Body is inserted in the suitable carrier for expression to express.When producing the expression vector, the coded sequence is located in the carrier, this Sample causes the suitable control sequence that the coded sequence is used to express with this to be operably connected.
Recombinant expression carrier can be can advantageously be subjected to recombinant DNA program and can cause polynucleotides express it is any Carrier (for example, plasmid or virus).The selection of carrier will typically depend on the carrier with there is the host of the carrier to be introduced thin The compatibility of born of the same parents.The carrier can be linear or closure circular plasmids.
Carrier can be autonomously replicationg vector, i.e. as carrier existing for extrachromosomal entity, it is replicated independently of dyeing Body replicates, for example, plasmid, extra-chromosomal element, minichromosomes or artificial chromosome.The carrier, which can include, to be used to ensure self Any device replicated.Alternately, the carrier can be such carrier, when it is introduced into the host cell, be integrated Replicated into genome and together with the one or more chromosomes for wherein having incorporated it.In addition it is possible to use single load (these carriers or plasmid jointly comprise the gene to be introduced into host cell for body or plasmid or two or more carriers or plasmid STb gene in group) or transposons.
The carrier, which preferably comprises one or more, to be allowed easily to select transformed cells, transfectional cell, transducer cell etc. thin The selective key thing of born of the same parents.Selective key thing is such a gene, the product of the gene provide biocide resistance or Virus resistance, heavy metal resistance, auxotrophic prototrophy etc..
The example of bacillary selected marker is bacillus licheniformis or bacillus subtilis dal genes, or assigns antibiosis The mark of plain resistance (such as ampicillin, chloramphenicol, kanamycins, neomycin, spectinomycin or tetracyclin resistance).
Carrier preferably comprise allow vector integration into the genome of host cell or carrier in cell independently of gene The element of group autonomous replication.
For being incorporated into the host cell gene group, the carrier can rely on encode the polypeptide polynucleotide sequence or Person is used for any other element by homologous or non-homologous re-combination to the carrier in the genome.Alternately, should Carrier, which can include, to be used to instruct to be incorporated into one or more of host cell gene group chromosome by homologous recombination One or more exact positions other polynucleotides.In order to increase the possibility integrated in exact position, these integration Element should include sufficient amount of nucleic acid, such as 100 to 10,000 base-pair, 400 to 10,000 base-pair and 800 to 10,000 base-pair, the sequence identity that these base-pairs have height with corresponding target sequence are homologous heavy to improve The possibility of group.These integrated elements can be the homologous any sequence of the target sequence in the genome with host cell.In addition, These integrated elements can be non-coding polynucleotide or coded polynucleotide.On the other hand, the carrier can be by non-same Source recombination and integration is into the genome of host cell.
For autonomous replication, carrier, which may further include, enables the carrier independently multiple in the host cell discussed The replication orgin of system.Replication orgin can be any plasmid replicon of the mediation autonomous replication to be worked in cell.Term " replication orgin " or " plasmid replicon " means the polynucleotides for enabling plasmid or carrier to replicate in vivo.
The example of bacterial origin of replication be allow replicated in Escherichia coli pBR322 plasmid, pUC19, pACYC177, And pACYC184 replication orgin, and allow replicated in bacillus plasmid pUB110, pE194, pTA1060, And pAM β 1 replication orgin.
The more than one copy Insertion Into Host Cell of polynucleotides of the present invention can be increased the generation of polypeptide.Pass through At least one other copy of sequence is incorporated into host cell gene group or by comprising together with the polynucleotides Amplifiable selected marker can obtain the increased copy numbers of polynucleotides, wherein by appropriate selection Property reagent in the presence of culture cell can select the cell and thus of the copy through amplification comprising selected marker The other copy of the polynucleotides.
To build the program of recombinant expression carrier of the present invention it is the general of this area for connecting element described above Known to logical technical staff (see, e.g., Sambrook et al., 1989, see above).
Host cell
The invention further relates to recombinant host cell, these recombinant host cells include the polynucleotides of the present invention, the multinuclear Thuja acid is operably coupled to one or more control sequences, and one or more control sequences instruct the production of the polypeptide of the present invention It is raw.Construct including polynucleotides or carrier are introduced into host cell, so that the construct or carrier are maintained work For chromosomal integrant or as carrier outside the chromosome of autonomous replication, as noted earlier.Term " host cell " cover due to The spawn of the mutation that occurs in the reproduction process parental cell different from parental cell.The selection of host cell is in very great Cheng Depend on encoding gene and its source of the polypeptide on degree.
Host cell can have any cell for recombinating the polypeptide for producing the present invention, such as prokaryotic.
Prokaryotic host cell can be any Gram-positive or gramnegative bacterium.Gram-positive bacterium include but It is not limited to：Bacillus, fusobacterium, enterococcus spp, Geobacillus, lactobacillus, lactococcus, bacillus marinus Category, staphylococcus, streptococcus and streptomyces.Gramnegative bacterium includes but is not limited to：It is campylobacter, big Enterobacteria, Flavobacterium, Fusobacterium, Helicobacterium, mud Bacillus, eisseria, Pseudomonas, Salmonella, And Ureaplasma.
Bacterial host cell can be any bacillus cell, include but is not limited to：Alkaliphilic bacillus, solution starch It is bacillus, bacillus brevis, Bacillus circulans, Bacillus clausii, bacillus coagulans, bacillus firmus, bright Rotten bacillus, bacillus lentus, bacillus licheniformis, bacillus megaterium, bacillus pumilus, stearothermophilus gemma bar Bacterium, bacillus subtilis and Bacillus thuringiensis cell.
Bacterial host cell can also be any streptococcus cell, include but is not limited to：Streptococcus equisimilis, make purulence hammer Bacterium, streptococcus uberis and streptococcus equi subsp blast cells.
Bacterial host cell can also be any Streptomyces cell, include but is not limited to：Not streptomyces chromogenes, deinsectization chain Mould, streptomyces coelicolor, streptomyces griseus and shallow Streptomyces glaucoviolaceus cell.
DNA is introduced into bacillus cell and can be realized by following：Protoplast transformation is (see, for example, Chang And Cohen, 1979, Mol.Gen.Genet. [molecular genetics and genomics] 168:111-115), competent cell converts (see, e.g., Young and Spizizen, 1961, J.Bacteriol. [Bacteriologies] 81:823-829；Or Dubnau with And Davidoff-Abelson, 1971, J.Mol.Biol. [J. Mol. BioLs] 56:209-221), electroporation is (referring to example Such as, Shigekawa and Dower, 1988, Biotechniques [biotechnologys] 6:742-751) or engagement (see, e.g. Koehler and Thorne, 1987, J.Bacteriol. [Bacteriologies] 169:5271-5278).DNA is introduced into Escherichia coli It can be realized in cell by following：Protoplast transformation (see, e.g., Hanahan, 1983, J.Mol.Biol., [give birth to by molecule Thing magazine] 166:557-580) or electroporation is (see, e.g., Dower et al., 1988, Nucleic Acids Res. [cores Acid research] 16:6127-6145).DNA is introduced into Streptomyces cell and can be realized by following：Protoplast transformation, electricity Perforation is (see, for example, Gong et al., 2004, Folia Microbiol. [microorganism grand sight] (Praha (Prague)) 49: 399-405), engagement is (see, for example, Mazodier et al., 1989, J.Bacteriol. [Bacteriologies] 171:3583- 3585) or transduction is (see, for example, Burke et al., 2001, Proc.Natl.Acad.Sci.USA [PNASs] 98:6289-6294).DNA is introduced into pseudomonas cell and can be realized by following：Electroporation is (see, e.g., Choi Et al., 2006, J.Microbiol.Methods [micro-biological process magazines] 64:391-397) or engagement (see, e.g., Pinedo and Smets, 2005, Appl.Environ.Microbiol. [application and environmental microbiologies] 71:51-57).By DNA Being introduced into streptococcus cell can be realized by following：Natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect.Immun. [infection is with being immunized] 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios [microbiologies] 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Appl.Environ.Microbiol. [application and environmental microbiologies] 65:3800-3804) or engagement (referring to, For example, Clewell, 1981, Microbiol.Rev. [Microbis] 45:409-436).However, it is possible to use this area Known any method that DNA is introduced to host cell.
Production method
The invention further relates to the method for producing polypeptide of the present invention, this method includes：(a) in the bar for being beneficial to produce the polypeptide The recombinant host cell of the present invention is cultivated under part；And optionally, (b) reclaims the polypeptide.
These host cells are in being adapted for use with method as known in the art and producing the nutrient medium of the polypeptide Culture.For example, can by Shaking culture or laboratory or industrial fermentation tank middle and small scale or large scale fermentation (including Continuously, in batches, fed-batch or solid state fermentation) culture cell, the culture is in suitable culture medium and is allowing to express And/or carried out under conditions of isolated polypeptide.The culture is to use program as known in the art, in a kind of suitable nutrient medium Middle generation, the culture medium include carbon and nitrogen source and inorganic salts.Suitable culture medium can obtain from commercial supplier or can root Prepared according to disclosed composition (for example, in catalogue of American type culture collection).If polypeptide is secreted into the battalion Support in culture medium, then polypeptide can be directly reclaimed from culture medium.If polypeptide is not secreted, then it can be from cell pyrolysis liquid Reclaimed.
Using specificity the polypeptide can be detected for the methods known in the art of the polypeptide.These detection method bags Include but be not limited to, the use of specific antibody, the formation of enzyme product or the disappearance of zymolyte.It is, for example, possible to use enzymatic determination comes Determine the activity of the polypeptide.
Polypeptide can be reclaimed using methods known in the art.For example, (it can be included but is not limited to by conventional program Collection, centrifuge, filter, extract, be spray-dried, evaporate or precipitate) reclaim the polypeptide from the nutrient medium.In one aspect, Recovery includes the zymotic fluid of the polypeptide.
Can by a variety of method purified polypeptides known in the art, methods described include but is not limited to chromatography (for example, Ion-exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography and size exclusion chromatography method), electrophoresis method (example Such as, preparative isoelectric focusing), otherness dissolving (for example, ammonium sulfate precipitation), SDS-PAGE or extraction (see, e.g., Protein Purification [protein purification], editing Janson and Ryden, VCH Publishers, [VCH publishes public Department], New York, 1989), to obtain substantially pure polypeptide.
Example
The microfluidic device of example 1.
Use soft lithography (Duffy, D.；McDonald,J.；Schueller,O.；Whitesides,G.； Anal.Chem. [analytical chemistry] 1998,70,4974-4984) by being poly- (dimethyl siloxane) (PDMS) by passage imitation To manufacture microfluidic device.In short, pass through the ultraviolet of mask (Selba SA [Sai Erba companies], Wei Er Suvas of Switzerland) Expose (MJB3 contact mask aligners；Germany plus emerging Su Si Microtechnologies Inc. (SUSS MicroTec, Garching, )) and subsequent development (SU-8 developers Germany；Microchem Corp. (MicroChem Corp.)) silicon chip (French Ah Your SILTRONIX companies (SILTRONIX, Archamp, France) still) on manufacture SU-8 photoresists (Massachusetts Microchem Corp. (MicroChem Corp., Newton, MA) of newton) mould.By curing agent add PDMS substrates (184 silicone elastomer kits；Lyons, France Dow Corning Corporation (Dow Corning Corp., Lyon, France to final concentration 10% (v/v), mixing in)), and it is poured on mould up to 5mm depth.After about 12h is crosslinked at 65 DEG C, PDMS is peeled off from mould, the HARRIS UNI-CORE of input and output port 0.75mm diametersTMBiopsy stamping machine punching press (Pennsylvania's Heartfield electron microscope company (Electron Microscopy Sciences, Hatfield,PA)).Using pressurized nitrogen PDMS particles are removed from port.By the way that two parts are exposed into oxygen plasma (PLASMA PREPTMII plasma furnaces；German Ahmedabad applies Wa Erbahe Jia La instruments company limited by shares (GaLa Instrumente GmbH, Bad Schwalbach, Germany)) and press them together, by the structuring of PDMS slabs Side is attached to 76 × 26 × 1mm glass microscope slide (the Borrow Malin Fei Er of Lauda, Germany-Konigshofen Moral company (Paul Marienfeld GmbH＆Co.KG, German on)).Finally, dredged with business Aqueous surface coating agent (The Pittsburg glassmaking company of Pennsylvania Pittsburg (Pittsburgh Glassworks Industries)) coating unit, is then rinsed with N2.Inject/take using normal pressure Go out the syringe pumps of PHD 22/2000 (German Nie Moxisi Cetoni companies (Cetoni, Nemesis, Germany)) by liquid pump Enter in microfluidic device.
The formation of the emulsion of example 2.
Emulsion can produce from many suitable combinations of immiscible liquid.The emulsion of the present invention has with separator well Drop form existing for aqueous phase (water containing biochemistry and biological components) and as suspending drops in carrier therein The commercially available hydrophobicity of fluid, immiscible liquid (contain 2% (w/w) PICO-SURFTMSurfactant (Britain Camb Spheroid fluidics company (Sphere Fluidics, Cambridge, UK)) HFE-7500 fluorinated oils).Such emulsion claims For Water-In-Oil (W/O).Emulsion is by the presence of surfactant and stable.
The generation of emulsion needs to apply mechanical energy to force two to be combined together.Exist various using different The method of mechanical device can do this part thing.We have used the microfluidic device or chip of referred to as droplet generator, its Force two to communicate meticulous nozzle, thus produce the single dispersing drop of Water-In-Oil.Such generator can be when very short Interior to produce substantial amounts of drop, its speed measures in the range of kHz.
Form the drop of the chloramphenicol growth medium containing Luria Bertani (LB), growth medium supplement There is the solution that expression encodes the B. subtilis cell of the gene library of a variety of amylase variants.Cell is diluted so that system Meter is packaged into often on learning according to Poisson distribution (Poisson distribution), no more than one B. subtilis cell In individual drop.The cell concentration used in this experiment only allows about 34% drop to be occupied by (single) bacterium, leaves emulsion In close to 66% drop do not have any living cells.
Total aqueous phase (bacterial cell with low concentration) is loaded into PTFE tube and (0.56 × 1.07mm inside/outsides footpath, flies generation that Bioblock scientific ＆ technical corporation (Fisher Bioblock Scientific)) in, and connect the tubing on syringe.Use injection Aqueous phase is expelled in droplet generator microfluidic device by pump with 100 μ L/h flow velocity.Gained liquid is passed through with 250 μ L/h flow velocity Flow with the flow focusing of above-mentioned carrier fluid to produce drop.It is 5kHz that drop, which produces speed,.Therefore, about 200.000 are encapsulated To provide about 600.000 drops, it is (wherein most that each drop statistically includes an at most individual cells in the library of bacterium Number drop is empty).Gained emulsion is stored in syringe 8 hours to allow bacterial growth and allow in drop at 37 DEG C Amylase produce and secretion.
Example 3. injects amylase substrate to detect enzymatic activity
After emulsion is incubated overnight, it is expelled in another microfluidic device again with 20 μ L/h speed, drop It is spaced apart with the carrier fluid injected with 120 μ L/h.Into each drop, addition small size (about 20% droplet size) can Commercially available fluorescence amylase substrate (ENZCHEKTMDetermination of amylase kit E33651, Life Technologies, Inc. (Life Technologies)).Continuous substrate is mutually injected/is fused to so that substrate to be added in drop in drop by voltolisation knot, with Realize the ultimate density of the about 25 μ g/L substrates in each drop in the PBS that pH is 7.By using model 623b high pressures Amplifier (Te Ruike companies (Trek, Inc.)；BF:OPTILAS SAS, Britain Camb) apply 600 volts under 30kHz between electrode Spy's exchange (AC) electric field, drop are merged with per second about 2 with substrate phase, and the speed of 000 fusion event is carried out 30 minutes.For 200.000 initial cells in 600.000 drops each provide substrate, and all drops are collected in syringe again In, then it is incubated 30 minutes at room temperature, so that amylase and the substrate reactions in drop.
Example 4. cracks selection amylase variant by cell
Drop incubation after, emulsion is expelled in another microfluidic device again with 20 μ L/h, and with 120 μ L/h Carrier fluid be spaced apart.When laser beam of the drop by 488nm excitation wavelengths, the fluorescence of all 600.000 drops is used Photomultiplier (PMT) measures.Do not show that the drop of fluorescence signal (therefore shows not have in drop living cells or only point Secrete the cell of nonactive amylase variant) lethal KOH/EDTA solution is injected using electric Agglomeration methods same as described above. Depending on the fluorescence signal detected from each drop, electric field is opened and closed.By the molten of 400mM KOH and 10mM EDTA Liquid is expelled in these negative drops, to realize 200mM KOH and 5mM EDTA ultimate density.The KOH of injection is in negative fluid Quickly mixed in drop, exist side by side and dissolve any cell therein, and make any protein or nucleic acid denaturation, so that negative fluid The content of drop prepares to be dropped.On the other hand, the positive drop of fluorescence signal is shownNoIt is injected lethal solution.It is positive Can also physical separation and separation from negative drop, then collect, but in this example, we are simply by lethal injection To cancel the negative drop of selection.
Cancel select after, we by add 10 μ L PICO-BREAKTM(the spheroid fluidics of Britain Camb is public Department (Sphere Fluidics, Cambridge, UK)) and 10mL fresh growth mediums destroy emulsion.From positive liquid The remaining bacillus cell of drop is not influenceed by the KOH from the drop for cancelling selection and EDTA, because gathering in all drops After knot, KOH/EDTA concentration has been diluted million times.Resulting solution (Innova 400, is flown into generation that at 37 DEG C with 220rpm Scientific ＆ technical corporation (Fisher scientist)) oscillation incubation is overnight to allow bacterial growth.
The selection that example 5. passes through PCR
As alternative choosing principles, we also carry out Direct PCR in positive drop, as follows：Repeat example 2-3 journey Sequence, but in example 3 after the final incubation of drop, by emulsion be expelled to again in new microfluidic device with 20 μ L/h and with Contain 2%w/w with 120 μ L/hSurfactant (the spheroid fluidics company (Sphere in Cambridge Fluidics, Cambridge)) HFE-7500 fluorinated oils be spaced apart.When drop is by a 488nm laser spots, photoelectricity is used Multiplier tube (PMT) measures the fluorescence of each drop.Each positive to showing the fluorescence signal higher than specific predetermined background level Droplet injection PCR solution.We use the continuous phase of the PCR reaction solutions containing 10 times of concentrations, PCR reaction solutions use KAPA HiFi HotStart ReadyMix PCR kits (KK2606, Life Technologies, Inc. (Life technologies)) With the specific primer of each amylase gene amplification in 3 μM of concentration.Using electric field consecutive PCR is injected for each positive drop Phase.Afterwards, the primer and 1 times of PCR that each drop contains ultimate density 300nM amylase encoding gene specific amplification mix Thing.The negative drop of fluorescence signal does not inject PCR solution needed for not showing.The activation that is injected through of continuous phase PCR solution applies The pulses of 1000V High Level AC Voltages (AC) on the electrode of neighbouring continuous aqueous phase is carried out.Depending on being examined from each drop The fluorescence signal measured, open and close electric field.
After selection, reclaim emulsion and be placed in thermal cycler, performing PCR is entered to emulsion.PCR reactions are carried out at 95 DEG C 3min, then at 98 DEG C, 20s；65 DEG C, 20s；With 72 DEG C, under 1.5min carry out 30 circulation, final incubation step be at 72 DEG C Lower carry out 5min.
After thermal cycle, by adding 10 μ L's(the spheroid fluidics company of Britain Camb (Sphere fluidics, Cambridge, UK)) emulsion is destroyed and by droplet coalescence.Pass through the phase between aqueous phase and oil phase Separate and recover aqueous phase.Only show that the amylase of fluorescence signal and the drop with being injected PCR mixtures and primer encodes base Because being amplified.
The sequence of positive hit can be identified by any suitable standard method, such as by cloning and being sequenced gene Or directly surveyed by using the sequencing primer of system marks in so-called " (NextGeneration) of future generation " sequencing equipment Sequence gene, as example disclosed in WO 2012/019765.
The use of the primer of biotin labeling is probably favourable in PCR solution.This will allow to use for example with strepto- The chromatographic column of Avidin rapidly and easily reclaims the PCR primer of amplification.
The microfluid of example 6. is equipped
Can use includes the microfluidic device of more than one syringe on same device so that drop interested can With from many different fluid rapid fusions.This will allow to sort the drop with positive signal into the storehouse of varying number, example Such as by intensity or its fluorescence return signal, i.e. enzymatic activity determines.This can be mixed by using the different PCR containing different primers Composition injection is dripped to carry out.Each storehouse can be defined by specific fluorescent strength range.This will allow based on different threshold values Drop selects.The selection in storehouse is by the level depending on the fluorescence signal from drop.Every group of primer in each syringe can be with Unique tags sequence with the storehouse for identifying quantity identical with syringe.For example, the device with three syringes will allow with Three fractions sort emulsion.By this way, each fraction can inject different groups of labeled primer to indicate that original host is thin The enzyme activity level (phenotype) of born of the same parents.

Claims (10)

1. a kind of method for being used for polynucleotides of identification code enzyme interested in microfluidic devices, methods described include with Lower step：
A) emulsion of the microfluid drop in the library comprising the polynucleotides for encoding one or more enzymes is provided, wherein each drop At most single member comprising the library, and the libraries of the polynucleotides is expressed to produce one or more enzymes；
B) aliquot of the substrate comprising one or more enzymes is introduced into each drop, one or more of which is interested The presence of organized enzyme the substrate is changed into the product that can be screened；
C) the positive drop for screening product containing higher than predetermined threshold level is determined, the predetermined threshold level is in turn The negative drop is determined again；
D) aliquot of the concentration PCR solution comprising nucleotides, suitable archaeal dna polymerase and one group of PCR primer is introduced, it is permitted The polynucleotides PCR amplifications for encoding one or more enzyme interested are arrived to each positive determined in step (c) perhaps In drop；And/or the aliquot for the solution that DNA is denatured and enzyme denaturation and/or cell crack will be caused to introduce in step (c) In the negative drop of middle determination；
E) polynucleotides of clone or PCR amplification codings one or more enzyme interested from the positive drop；With
F) polynucleotides of the polynucleotides identification code of the clone from step (e) or PCR amplifications enzyme interested.
2. the method as described in claim 1, the wherein enzyme interested are hydrolase, isomerase, ligase, lyases, oxygen Change reductase or transferase；Preferably, the enzyme interested is a kind of alpha-galactosidase, alpha-Glucosidase, aminopeptidase, starch Enzyme, asparaginase, beta galactosidase, β-glucosyl enzym, xylobiase, carbohydrase, carboxypeptidase, catalase, fiber Disaccharide-hydrolysing enzymes, cellulase, chitinase, cutinase, cyclodextrin glycosyl transferases, deoxyribonuclease, inscribe Portugal gather Carbohydrase, esterase, green fluorescent protein, glucanotransferase, glucoamylase, invertase, laccase, lipase, mannosidase, Become dextranase, oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonucleic acid Enzyme, transglutaminase or zytase.
3. method as claimed in claim 1 or 2, the wherein library encode one or more hydrolases, isomerase, ligase, Lyases, oxidoreducing enzyme or transferase；Preferably, the library encode one or more alpha-galactosidases, alpha-Glucosidase, Aminopeptidase, amylase, asparaginase, beta galactosidase, β-glucosyl enzym, xylobiase, carbohydrase, carboxypeptidase, peroxide Change hydrogen enzyme, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyl transferases, DNA Enzyme, endoglucanase, esterase, green fluorescent protein, glucanotransferase, glucoamylase, invertase, laccase, lipase, Mannosidase, become dextranase, oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolysis Enzyme, ribalgilase, transglutaminase or zytase.
4. the method as any one of preceding claims, the wherein library encode the different variants of same enzyme.
5. the method as any one of preceding claims, the wherein library include two or more of coding same enzyme Different polynucleotides.
6. the method as any one of preceding claims, the wherein library are comprised in prokaryotic host cell；It is preferred that In gram positive host cell；More preferably in Bacillus host cell；Even more preferably in Alkaliphilic bacillus, solution Bacillus amyloliquefacienses, bacillus brevis, Bacillus circulans, Bacillus clausii, bacillus coagulans, hard gemma bar Bacterium, bacillus lautus, bacillus lentus, bacillus licheniformis, bacillus megaterium, bacillus pumilus, stearothermophilus In bacillus, bacillus subtilis or bacillus thuringiensis host cell；And most preferably in bacillus licheniformis host Into the cell.
7. method as claimed in claim 7, wherein each drop in step (a) includes at most single host cell.
8. the substrate of the method as any one of preceding claims, wherein one or more enzymes is fluorescence, and And the fluorogenic substrate is converted into fluorescence-causing substance by the activity of the wherein enzyme.
9. the method as any one of preceding claims, the polynucleotides of wherein coding enzyme interested are more by this The DNA sequencing of nucleotides is accredited in step (f).
10. the method as any one of preceding claims, the wherein aliquot are by applying electric field or passing through injection The aliquot and the droplet coalescence or coalescence are introduced into the drop.
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